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Vincent Geli (Instabilité du Génome Et Cancérogénèse, IGC, Marseille)

LES TELOMERES SONT MULTIFONCTIONNELSET CONTROLENT LA PROLIFERATION CELLULAIREET LA STABILITE DU GENOME

- PROTECTION DES EXTREMITES

-REPLICATION COMPLETE -DES CHROMOSOMES

- ORGANISATION DES CHROMOSOMES

cancerageing

DNA DNA damagedamage

Rif1

Rif2

Rif1 Rif1

TRF2TRF2TRF1 Pot1

TPP1

Taz1

Taz1 Taz1 Pot1 Pot1

Rap1 Rap1 Rap1 Cdc13

Ten1

Rif1

Rif2

Rif1

swi6

SirSir

HP1

Rap1 Rap1 Rap1

RAP1RAP1

TRF1Tin2 Tin2

HP1HP1

Pot1

TPP1

Pot1

TPP1

Pot1

TPP1

Ciliate

Buddingyeast

Fissionyeast

Mammals

Stn1

3’

3’

3’

3’

E. Gilson and V. Géli, 2007.

Titia de Lange, 2004, 2005

• Repeated sequences at the end of linear chromosomes (TTAGGG in humans)

• Protects against environmental damage, recombination with other chromosomes, & from loss of information during replication

Forms a guanine-quadraduplex

Dysfunctional telomeres

Genomic instability

The Eukaryotic Problem of Telomere Replication

RNA primer near end of the chromosome on lagging strand can’t be replaced with DNA since DNA polymerase must add to a primer sequence.

Do chromosomes get shorter with each replication?

Replication of the ends of linear DNA

?

5'

3'

Eukaryotes — TELOMERES and TELOMERASE

Telomeric DNA consists of TANDEM REPEATS of simple sequences:

Tetrahymena TTGGGG

Humans TTAGGG 5-15 kbp

D. Termination

3’

Telomeric DNA is synthesized by the semi-conservative replication machinery

3’

3’

3’

Gradual telomere shortening

Unprotected telomere

Apoptosis Senescence

Dynamique de la taille des télomères pendant le développement

BIRTHDEATHIn germinal cellsTelomerase activity

Telomere length

Telomerase activity

Telomere length

In somatic cells

La télomérase est régulée au cours du développement

Cellules germinales

Cellules somatiques

Naissance Mort

Taille des télomères

Télomérase

Taille des télomères

Télomérase

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cette image.

Structure des télomères

temps

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Diapositive 23Diapositive 23

Permanent arrest = senescence

Proliferation of primary normal human cells is not indefinite 1. period of rapid proliferation 2. slowing of the proliferation rate 3. cessation of proliferation

= replicative senescence

Senescent cells permanently arrested remains viable for extended periods of time changes in morphology, nuclear structure gene expression, metabolism

The role of senescence :a limit to proliferation

Suggested purpose : to limit the proliferative capacity of normal cells

-> a mechanism involved in normal development & tissue maintenance

-> a potent tumor-protection mechanism

-> must be prevented in proliferative cell compartments(stem cells)

Sénescence(M1)

Crise(M2)

Immortalité

P53RB

hTERT+++

RandomDNA break

Anaphase

Telomere dysfunctioncan initiate genome instability

Repeated generation of DNA breaksthrough breakage-fusion-bridge cycles

End-to-endfusion

Telomere length dynamics during malignant transformation

Telomerase activity

Telomere

length

Extensive proliferationAbnormalproliferation

90 % des cancers surexpriment la télomérase

5 % des cancers ne surexpriment pas la télomérasemais activent une voie alternative du maintien de l’ADN télomérique

= ALT

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3’

Most telomeric DNA is synthesized by the semi-conservative replication machinery

Telomerase

3’

3’

3’

Gradual telomere shortening

(only 7% of cell cycles)

THE TELOMERASE

The telomerase complex

AACCCCAAC3' 5'RNA

AACCCCAAC3' 5'RNA

TELOMERASE

These repeat sequences are found as 3' overhangs:

5'TTGGGGTTGGGGTTGGGG 3'3'AACCCC 5'

(TTGGGG)n

5'TTGGGGTTGGGGTTGGGG 3'AACCCC 5'

3'TGT

5'TTGGGGTTGGGGTTGGGGTTG 3'AACCCC 5'

GGGTTG

Translocation

How is telomerase recruited to the telomere?

How is telomerase action coupled to DNA replication?

What tell us the yeast system model

Rif1Rif2

Rap1 Rap1 Rap1Cdc13

Ten1

Rif1

Rif2

SirSir

Buddingyeast

Stn1

3’

E. Gilson and V. Géli, 2007.

Est 2TLC1

ADNPol

ADNPol

RFC

RPA

5’3’

5’3’

FEN-1

DNA ligase 1

Rnase H1

PCNA

Primase

Helicase

Okasaki fragments

5’

3’

Cdc13-Stn1-Ten1

Rap1 Rap1 Ku

Late S-phase

N. Hug and J. LingnerChromosoma, june 2006

Est1-Myc

Est2-Myc

Cdc13-Myc

yKu80-Myc

0

10

20

30

40

50

60

0 15 30 45 60 75 Time (min)

Fo

ld e

nric

hm

en

t(T

el/B

ack

gro

un

d) wt

Ku

Ku

Est 2Est1

Est 3

5’3’

3’

5’Cdc13

Telomerase is acting in S-phase (E. Gilson, D. Gottschling)

Telomerase action requires fork passage (E. Gilson, D. Gottschling)

Generation of ssDNA G-tails is required for telomerase action(R. Wellinger)

G1

Early S Late S

M

Ten1

Est 2

TLC1

KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Ten1

Leading strand

Lagging strand

Rap1

Rap1 Rap1 Ku Cdc13

Stn1

3’

Est 2Rap1

G1

Early S Late S

S/G2

M

Est3Est1

TLC1

Est2Cdc13

RPA

Rap1

Ku ?

Cdk1

?

TLC1

Est3

Tel1

Cdc13Est2

Rap1

MRXExo?

Cdk1

?

Lagging telomerase Leading telomeraseKu ?

Ten1

Leading strand

Lagging strand

Rap1

Rap1 Rap1 Ku Cdc13

Stn1

3’

Est 2Rap1

Est1

Telomerase activation is linked to the passage of the replication fork

G1

Early S Late S

S/G2

M Est3

Est2

TLC1

Pol/Pol12Primase

Est1

Pif1

RPA

Rap1 Rap1 Rap1

Rif1Rif2

Cdc13 Cdc13Rap1 Rap1

Est3Est1

TLC1

RPA

Est2Cdc13

RPA

Rap1

Ku ?

Cdk1

?

TLC1

Est3

RPATel1

Cdc13Est2

Rap1

MRXExo?

Cdk1

?

Lagging telomerase Leading telomeraseKu ?

Est1

G1

Early S Late S

S/G2

M

Ten1

Est 2

TLC1

KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Ten1KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Est3

Est2

TLC1

Pol/Pol12Primase

Est1

Pif1

RPA

Rap1 Rap1 Rap1

Rif1Rif2

Cdc13 Cdc13Rap1 Rap1

G1

Early S Late S

S/G2

M

Ten1

Est 2

TLC1

KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Ten1KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Est3

Est2

TLC1

Pol/Pol12Primase

Est1

Pif1

RPA

Rap1 Rap1 Rap1

Rif1Rif2

Cdc13 Cdc13Rap1 Rap1

Est3Est1

TLC1

Est2Cdc13

RPA

Rap1

Ku ?

Cdk1

?

TLC1

Est3

Tel1

Cdc13Est2

Rap1

MRXExo?

Cdk1

?

Lagging telomerase Leading telomeraseKu ?

Ten1

Leading strand

Lagging strand

Rap1

Rap1 Rap1 Ku Cdc13

Stn1

3’

Est 2Rap1

Est1

E. Gilson and V. Géli, 2007

The telomerase cycle

G1

Early S Late S

S/G2

M

Ten1

Est 2

TLC1

KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Ten1KuRap1 Rap1 Rap1 Cdc13

Stn1

Ten1

Rap1 3’

Rif1Rif2 Rif1

Rif2 Rif1Rif2

Sir

Est3

Est2

TLC1

Pol/Pol12Primase

Est1

Pif1

RPA

Rap1 Rap1 Rap1

Rif1Rif2

Cdc13 Cdc13Rap1 Rap1

Est3Est1

TLC1

RPA

Est2Cdc13

RPA

Rap1

Ku ?

Cdk1

?

TLC1

Est3

RPATel1

Cdc13Est2

Rap1

MRXExo?

Cdk1

?

Lagging telomerase Leading telomeraseKu ?

Ten1

Leading strand

Lagging strand

Rap1

Rap1 Rap1 Ku Cdc13

Stn1

3’

Est 2Rap1

Est1

E. Gilson and V. Géli, 2007